益肾养髓方对脊髓型颈椎病大鼠脊髓中神经营养因子表达的影响及其神经修复作用研究

背景　骨关节退行性改变压迫脊髓所致的脊髓型颈椎病（CSM）与脊髓急性损伤在病理特点及治疗策略方面存在差异，中医药治疗退行性CSM有着独特优势，临床应用广泛，但脊髓慢性压迫相关的治疗机制研究较少。目的　研究益肾养髓方对脊髓慢性压迫大鼠脊髓（C5~7）神经生长因子（NGF）、神经营养因子3（NT3）、胶质细胞源性神经营养因子（GDNF）的mRNA表达水平影响及NGF的蛋白表达和分布情况，探讨益肾养髓方促进CSM神经功能修复的作用机制。方法　2019年1月选取SPF级雌性SD大鼠96只，其中72只大鼠采用吸水膨胀材料聚乙烯醇丙烯酰胺互穿网络水凝胶（由北京师范大学化学学院提供）压迫脊髓的方法进行造模，余24只大鼠假手术（假手术组）。造模成功的大鼠随机分为模型组（n=24）、中药高浓度组（n=16）、中药中浓度组（n=16）、中药低浓度组（n=16）。中药灌胃的各组按照对应浓度进行灌胃（大鼠灌胃给药剂量采用体表面积系数换算系数6.3计算，高浓度为16.74 g•kg-1•d-1、中浓度为8.37 g•kg-1•d-1、低浓度为4.19 g•kg-1•d-1），药液浓缩后以1 ml/100 g体积灌胃给药，模型组及假手术组灌服等量（1 ml/100 g）0.9%氯化钠注射液，1次/d。分别于术后2、4、6、8、10周时，评价各组大鼠运动功能指数评分（BBB评分）；术后2、6、10周时，尼氏染色法观察大鼠脊髓前角正常神经元的尼氏体分布，实时荧光定量逆转录聚合酶链式反应（RT-qPCR） 法检测大鼠脊髓营养因子NGF、NT3、GDNF的 mRNA表达水平、免疫组织化学分析NGF脊髓前角区蛋白表达及分布情况。结果　（1）术后2周，假手术组BBB评分高于模型组、中药高浓度组、中药中浓度组和中药低浓度组（P<0.05）；术后4周，假手术组BBB评分高于模型组、中药高浓度组、中药中浓度组和中药低浓度组（P<0.05）；术后6周，假手术组BBB评分高于模型组、中药高浓度组、中药中浓度组和中药低浓度组（P<0.05），模型组BBB评分低于中药高浓度组（P<0.05），中药高浓度组BBB评分高于中药中、低浓度组（P<0.05）；术后8周，假手术组BBB评分高于模型组、中药高浓度组、中药中浓度组和中药低浓度组（P<0.05），模型组BBB评分低于中药高浓度组（P<0.05），中药高浓度组BBB评分高于中药中、低浓度组（P<0.05）；术后10周，假手术组BBB评分高于模型组、中药高浓度组、中药中浓度组和中药低浓度组（P<0.05），模型组BBB评分低于中药高、中浓度组（P<0.05），中药高浓度组BBB评分高于中药中、低浓度组（P<0.05）。（2）术后2周，假手术组脊髓前角运动神经元形态正常，可见大量虎斑状尼氏体，丰富饱满；模型组脊髓前角神经元细小，形态为圆形，分布稀疏，胞内尼氏体溶解减少甚至消失；在术后6、10周时，中药高、中浓度组神经元虽有一定程度损伤，但细胞形态丰满、可见细胞内虎斑状尼氏体；中药低浓度组可见少量空泡，神经元细胞分布略稀疏，单个细胞内尼氏体数目比中药高、中浓度组含量少。术后2周，假手术组正常神经元数目多于模型组（P<0.05）。术后6周，假手术组正常神经元数目多于模型组、中药中浓度组和中药低浓度组（P<0.05），模型组正常神经元数目少于中药高浓度组（P<0.05），中药高浓度组正常神经元数目多于中药低浓度组（P<0.05）。术后10周，假手术组正常神经元数目多于模型组、中药低浓度组（P<0.05），模型组正常神经元数目少于中药高浓度组（P<0.05）。（3）术后6周，中药高浓度组NGF mRNA表达水平高于假手术组、模型组和中药低浓度组（P<0.05），中药高浓度组NT3 mRNA表达水平高于模型组（P<0.05），中药高浓度组GDNF mRNA表达水平高于假手术组、模型组和中药中、低浓度组（P<0.05）。（4）假手术组和模型组中脊髓前角的神经元细胞NGF染色程度较浅，分布稀疏，中药高、中浓度组大鼠的脊髓前角神经元NGF染色较明显，细胞形态完整。术后6周，中药高浓度组NGF平均积分光密度值高于假手术组和模型组（P<0.05）。结论　益肾养髓方可能通过增加脊髓中NGF、NT3、GDNF的mRNA表达水平，改善CSM大鼠的四肢运动功能，保护脊髓前角中正常运动神经元，从而达到促进神经修复的效果。

Effect of Yishen Yangsui Formula on Expression of Neurotrophic Factorsanditsnerve Repair Effect in Spinal Cord of Rats with Cervical Spondylotic Myelopathy

Background　There are differences in pathological characteristics and treatment strategies between acute spinal cord injury（ASCI） and cervical spondylotic myelopathy（CSM） by chronic compression. Traditional Chinese Medicine （TCM） has unique advantages in the treatment of CSM with extensive clinical application，but there were few studies on the treatment mechanism related to chronic compression of spinal cord. Objective　To study the effect of Yishen Yangsui formula Chinese medicine on the mRNA expression levels of nerve growth factor（NGF），neurotrophic factor 3（NT3），glial cell derived neurotrophic factor（GDNF） and the protein expression and distribution of NGF in the spinal cord of chronic spinal cord compression rats（C5-7），in order to explore the mechanism of TCM promoting nerve repair in CSM. Methods　A total of 96 SPF female SD rats were selected，of which 72 rats were operated by spinal cord compression with water absorbent swelling material polyvinyl alcohol acrylamide in terpenetrating network hydrogel（provided by College of Chemistry，Beijing Normal University），and the remaining 24 rats were operated under sham operation（sham operation group）. The successfully modeled rats were divided into model group（n=24），high concentration herb formula group（n=16），medium concentration herb formula group（n=16），low concentration herb formula group（n=16）. Each group was given intragastric administration of herb formula according to the corresponding concentration（the dose of intragastric administration of rats was calculated by the conversion coefficient of body surface area coefficient 6.3，high concentration was 16.74，medium concentration was 8.37 g•kg-1•d-1 and low concentration was 4.19 g•kg-1•d-1）. After the liquid was concentrated，it was given by intragastric administration in a volume of 1 ml/100 g. The model group and the sham operation group were given the same amount（1 ml/100 g）of 0.9% sodium chloride intragastric administration once daily. BBB scores of rats in each group were evaluated at 2，4，6，8 and 10 weeks postoperatively. At 2 weeks，6 weeks and 10 weeks after surgery，the distribution of normal nerve cells in the ventral horn of the spinal cord of rats was observed by Nissl staining. The mRNA of NGF，NT3，GDNF was detected by RT-qPCR，the protein expression and distribution in the anterior horn region of the spinal cord of NGF were analyzed by immunohistochemistry. Results　（1）At 2 weeks after operation，BBB score of sham operation group was higher than model group（P<0. 05）；At 4 weeks after operation，BBB score of sham operation group was higher than model group，high concentration herb formula group，medium concentration herb formula group and low concentration herb formula group（P<0.05）；at 6 weeks after operation，BBB score of sham operation group was higher than model group，high concentration herb formula group，medium concentration herb formula group and low concentration herb formula group（P<0.05）；BBB score of model group was lower than that of high concentration herb formula group（P<0.05）；BBB score of high concentration herb formula group was higher than medium concentration herb formula group and low concentration group（P<0.05）；at 8 weeks after operation，BBB score of sham operation group was higher than model group，high concentration herb formula group，medium concentration herb formula group and low concentration herb formula group（P<0.05）；BBB score of model group was lower than high concentration herb formula group（P<0.05）；BBB score of high concentration group was higher than medium concentration herb formula group and low concentration herb formula group（P<0.05）；at 10 weeks after operation，BBB score of sham operation group was higher than model group，high concentration herb formula group，medium concentration herb formula group and low concentration herb formula group（P<0.05）；BBB score of model group was lower than high and medium herb formula concentration group（P<0.05）；BBB score of high concentration herb formula group was higher than low concentration herb formula group（P<0.05）. （2）At 2 weeks after operation，in the sham operation group，the motor neurons in the anterior horn of the spinal cord were normal in shape，with abundant Nissl bodies. In the model group，the neurons in the anterior horn of the spinal cord were small，round and sparsely distributed，and the intracellular Nissl bodies decreased or even disappeared. At 6 and 10 weeks after operation，the neurons in the group of high and medium concentration of herb formula were damaged to a certain extent，but the cell morphology was plump and intracellular Nissl bodies was visible. A small number of vacuoles were observed in the low concentration herb formula group，and the distribution of neuron cells was slightly sparse. The normal cell with Nissl bodies of low concentration herb formula group was less than the high and medium concentration herb formula group（P<0.05）. At 2 weeks after the operation，the number of normal cells in the sham operation group was more than that in the model group（P<0.05）. At 6 weeks after operation，the normal cells in the sham operation group was more than that in the model group，medium concentration herb formula group and low concentration herb formula group（P<0.05）；the normal cell in the model group was less than that in the high herb formula concentration group（P<0.05）；the normal cells in high concentration herb formula group was higher than that in low herb formula concentration group（P<0.05）. At 10 weeks after operation，the normal cells in sham operation group was higher than that in model group and low concentration herb formula group（P<0. 05）；The normal cell in model group was lower than that of high concentration herb formula group（P<0.05）. （3）At 6 weeks after operation，NGF mRNA in high concentration herb formula group was higher than sham operation group，model group and low concentration herb formula group（P<0.05）. NT3 mRNA in high concentration herb formula group was higher than model group（P<0.05）. GDNF mRNA in high herb formula concentration group was higher than sham operation group，model group，medium and low concentration herb formula groups（P<0.05）. （4）The NGF staining of neurons in the anterior horn of spinal cord in the sham operation group and the model group was shallow and sparsely distributed，while the NGF staining of neurons in the anterior horn of spinal cord in the high and medium herb formula concentration groups was obvious and the cell morphology was intact. At 6 weeks after operation，the average integral optical density of NGF in the high herb formula concentration group was higher than that in the sham group and the model group（P<0.05）. Conclusion　By increasing the expression levels of NGF，NT3 and GDNF mRNA in the spinal cord，the Yishen Yangsui formula may improve the limb motor function of the rats with CSM，maintain the number of normal motor neurons in the anterior horn of the spinal cord，and achieve the effect of promoting nerve repair.